scholarly journals Deletion of the min Operon Results in Increased Thermosensitivity of an ftsZ84 Mutant and Abnormal FtsZ Ring Assembly, Placement, and Disassembly

2000 ◽  
Vol 182 (21) ◽  
pp. 6203-6213 ◽  
Author(s):  
Xuan-Chuan Yu ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
High Frequency ◽  
Double Mutant ◽  
Nonpermissive Temperature ◽  
Ftsz Ring ◽  
Single Mutant ◽  
Ring Assembly ◽  
Z Ring ◽  
Thermosensitive Mutation

ABSTRACT To investigate the interaction between FtsZ and the Min system during cell division of Escherichia coli, we examined the effects of combining a well-known thermosensitive mutation offtsZ, ftsZ84, with ΔminCDE, a deletion of the entire min locus. Because the Min system is thought to down-regulate Z-ring assembly, the prediction was that removing minCDE might at least partially suppress the thermosensitivity of ftsZ84, which can form colonies below 42°C but not at or above 42°C. Contrary to expectations, the double mutant was significantly more thermosensitive than theftsZ84 single mutant. When shifted to the new lower nonpermissive temperature, the double mutant formed long filaments mostly devoid of Z rings, suggesting a likely cause of the increased thermosensitivity. Interestingly, even at 22°C, many Z rings were missing in the double mutant, and the rings that were present were predominantly at the cell poles. Of these, a large number were present only at one pole. These cells exhibited a higher than expected incidence of polar divisions, with a bias toward the newest pole. Moreover, some cells exhibited dramatically elongated septa that stained for FtsZ, suggesting that the double mutant is defective in Z-ring disassembly, and providing a possible mechanism for the polar bias. Thermoresistant suppressors of the double mutant arose that had modestly increased levels of FtsZ84. These cells also exhibited elongated septa and, in addition, produced a high frequency of branched cells. A thermoresistant suppressor of the ftsZ84 single mutant also synthesized more FtsZ84 and produced branched cells. The evidence from this study indicates that removing the Min system exposes and exacerbates the inherent defects of the FtsZ84 protein, resulting in clear septation phenotypes even at low growth temperatures. Increasing levels of FtsZ84 can suppress some, but not all, of these phenotypes.

scholarly journals A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

2021 ◽  
Author(s):  
Shirin Ansari ◽  
James C. Walsh ◽  
Amy L. Bottomley ◽  
Iain G. Duggin ◽  
Catherine Burke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Sos Response ◽  
E Coli ◽  
Ring Assembly ◽  
Multiple Alternative ◽  
Z Ring ◽  
Cell Division Inhibitor

Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple alternative division inhibition pathways exist during the SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism. Importance: Filamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

scholarly journals Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ

2000 ◽  
Vol 182 (14) ◽  
pp. 3965-3971 ◽  
Author(s):  
Zonglin Hu ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Functional Domains ◽  
Wild Type ◽  
Terminal Domain ◽  
Ring Assembly ◽  
Z Ring ◽  
Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

scholarly journals The C-Terminal Domain of MinC Inhibits Assembly of the Z Ring in Escherichia coli

2006 ◽  
Vol 189 (1) ◽  
pp. 236-243 ◽  
Author(s):  
Daisuke Shiomi ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Additional Function ◽  
Ring Assembly ◽  
C Terminus ◽  
Min Proteins ◽  
Z Ring ◽  
Ftsz Assembly ◽  
Green Fluorescent

ABSTRACT In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC122-231) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC122-231, the C terminus of full-length MinC, or the C terminus of MinC122-231 perturbed MinC function, which may explain why cell division inhibition by MinC122-231 was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

scholarly journals A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

2020 ◽  
Author(s):  
Shirin Ansari ◽  
James C. Walsh ◽  
Amy L. Bottomley ◽  
Iain G. Duggin ◽  
Catherine Burke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Sos Response ◽  
E Coli ◽  
Ring Assembly ◽  
Z Ring ◽  
Response To Stress ◽  
Cell Division Inhibitor

AbstractRod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC+ phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple division inhibition pathways exist during the stress-induced SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism.ImportanceFilamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

scholarly journals Escherichia coli Division Inhibitor MinCD Blocks Septation by Preventing Z-Ring Formation

2001 ◽  
Vol 183 (22) ◽  
pp. 6630-6635 ◽  
Author(s):  
Sebastien Pichoff ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Direct Interaction ◽  
Ring Formation ◽  
Ring Assembly ◽  
Z Ring ◽  
Alternative Fixation ◽  
Green Fluorescent

ABSTRACT The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118–1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410–423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles offtsZ, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.

scholarly journals Role of FtsEX in Cell Division of Escherichia coli: Viability of ftsEX Mutants Is Dependent on Functional SufI or High Osmotic Strength

2006 ◽  
Vol 189 (1) ◽  
pp. 98-108 ◽  
Author(s):  
Manjula Reddy
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Salt Transport ◽  
High Osmolarity ◽  
Growth Defects ◽  
Ring Assembly ◽  
Multiple Copies ◽  
Z Ring ◽  
Twin Arginine Translocase ◽  
The Stability

ABSTRACT In Escherichia coli, at least 12 proteins, FtsZ, ZipA, FtsA, FtsE/X, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC, are known to localize to the septal ring in an interdependent and sequential pathway to coordinate the septum formation at the midcell. The FtsEX complex is the latest recruit of this pathway, and unlike other division proteins, it is shown to be essential only on low-salt media. In this study, it is shown that ftsEX null mutations are not only salt remedial but also osmoremedial, which suggests that FtsEX may not be involved in salt transport as previously thought. Increased coexpression of cell division proteins FtsQ-FtsA-FtsZ or FtsN alone restored the growth defects of ftsEX mutants. ftsEX deletion exacerbated the defects of most of the mutants affected in Z ring localization and septal assembly; however, the ftsZ84 allele was a weak suppressor of ftsEX. The viability of ftsEX mutants in high-osmolarity conditions was shown to be dependent on the presence of a periplasmic protein, SufI, a substrate of twin-arginine translocase. In addition, SufI in multiple copies could substitute for the functions of FtsEX. Taken together, these results suggest that FtsE and FtsX are absolutely required for the process of cell division in conditions of low osmotic strength for the stability of the septal ring assembly and that, during high-osmolarity conditions, the FtsEX and SufI functions are redundant for this essential process.

scholarly journals MinC N- and C-Domain Interactions Modulate FtsZ Assembly, Division Site Selection, and MinD-Dependent Oscillation inEscherichia coli

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Christopher J. LaBreck ◽  
Joseph Conti ◽  
Marissa G. Viola ◽  
Jodi L. Camberg
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Site Selection ◽  
Fluorescent Protein ◽  
Site Mutation ◽  
Content Type ◽  
Ftsz Ring ◽  
Division Site ◽  
Z Ring

ABSTRACTThe Min system inEscherichia coli, consisting of MinC, MinD, and MinE proteins, regulates division site selection by preventing assembly of the FtsZ-ring (Z-ring) and exhibits polar oscillationin vivo. MinC antagonizes FtsZ polymerization, andin vivo, the cellular location of MinC is controlled by a direct association with MinD at the membrane. To further understand the interactions of MinC with FtsZ and MinD, we performed a mutagenesis screen to identify substitutions inminCthat are associated with defects in cell division. We identified amino acids in both the N- and C-domains of MinC that are important for direct interactions with FtsZ and MinDin vitro, as well as mutations that modify the observedin vivooscillation of green fluorescent protein (GFP)-MinC. Our results indicate that there are two distinct surface-exposed sites on MinC that are important for direct interactions with FtsZ, one at a cleft on the surface of the N-domain and a second on the C-domain that is adjacent to the MinD interaction site. Mutation of either of these sites leads to slower oscillation of GFP-MinCin vivo, although the MinC mutant proteins are still capable of a direct interaction with MinD in phospholipid recruitment assays. Furthermore, we demonstrate that interactions between FtsZ and both sites of MinC identified here are important for assembly of FtsZ-MinC-MinD complexes and that the conserved C-terminal end of FtsZ is not required for MinC-MinD complex formation with GTP-dependent FtsZ polymers.IMPORTANCEBacterial cell division proceeds through the coordinated assembly of the FtsZ-ring, or Z-ring, at the site of division. Assembly of the Z-ring requires polymerization of FtsZ, which is regulated by several proteins in the cell. InEscherichia coli, the Min system, which contains MinC, MinD, and MinE proteins, exhibits polar oscillation and inhibits the assembly of FtsZ at nonseptal locations. Here, we identify regions on the surface of MinC that are important for contacting FtsZ and destabilizing FtsZ polymers.

scholarly journals New Temperature-Sensitive Alleles of ftsZ in Escherichia coli

2005 ◽  
Vol 187 (1) ◽  
pp. 358-365 ◽  
Author(s):  
Stephen G. Addinall ◽  
Elaine Small ◽  
Duncan Whitaker ◽  
Shane Sturrock ◽  
William D. Donachie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Point Mutations ◽  
Ring Formation ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Z Ring

ABSTRACT We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli, using P1-mediated, localized mutagenesis. The five resulting single amino acid changes (Gly109→Ser109 for ftsZ6460, Ala129→Thr129 for ftsZ972, Val157→Met157 for ftsZ2066, Pro203→Leu203 for ftsZ9124, and Ala239→Val239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42°C. In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42°C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30°C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature. Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran. Hence, both mutations also cause defects in FtsZ polymerization in vitro. Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein. We demonstrate here that unlike expression of ftsZ84, multicopy expression of the ftsZ6460, ftsZ972, and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature. In addition, all five new mutant FtsZ proteins are stable at 42°C. Therefore, the novel isolates carrying single ftsZ(Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.

scholarly journals Aberrant Cell Division and Random FtsZ Ring Positioning in Escherichia coli cpxA* Mutants

1998 ◽  
Vol 180 (13) ◽  
pp. 3486-3490 ◽  
Author(s):  
Joe Pogliano ◽  
Jian Ming Dong ◽  
Peter De Wulf ◽  
Dierdre Furlong ◽  
Dana Boyd ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Sensor Kinase ◽  
Response Regulators ◽  
Gene Encoding ◽  
Ftsz Ring ◽  
Division Site ◽  
Ring Assembly ◽  
Sensor Kinases ◽  
Two Component Signal Transduction

ABSTRACT In Escherichia coli, certain mutations in thecpxA gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division. However, deletion of the cpxRA operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis. It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division.

scholarly journals Timing of FtsZ Assembly in Escherichia coli

1999 ◽  
Vol 181 (17) ◽  
pp. 5167-5175 ◽  
Author(s):  
Tanneke Den Blaauwen ◽  
Nienke Buddelmeijer ◽  
Mirjam E. G. Aarsman ◽  
Cor M. Hameete ◽  
Nanne Nanninga
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Dna Replication ◽  
Growth Conditions ◽  
Ftsz Ring ◽  
Contrast Microscopy ◽  
Z Ring ◽  
Ftsz Assembly ◽  
K 12

ABSTRACT The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.

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